Increased TLR4 expression in murine placentas after oral infection with periodontal pathogens

Abstract

Maternal periodontitis has emerged as a putative risk factor for preterm births in humans. The periodontitis-associated dental biofilm is thought to serve as an important source of oral bacteria and related virulence factors that hematogenously disseminate and affect the fetoplacental unit; however the underlying biological mechanisms are yet to be fully elucidated. This study hypothesized that an oral infection with the human periodontal pathogens Campylobacter rectus and Porphyromonas gingivalis is able to induce fetal growth restriction, placental inflammation and enhance Toll-like receptors type 4 (TLR4) expression in a murine pregnancy model. Female Balb/C mice (n = 40) were orally infected with C. rectus and/or P. gingivalis over a 16-week period and mated once/week. Pregnant mice were sacrificed at embryonic day (E) 16.5 and placentas were collected and analyzed for TLR4 mRNA levels and qualitative protein expression by real-time PCR and immunofluorescence. TLR4 mRNA expression was found to be increased in the C. rectus-infected group (1.98 +/- 0.886-fold difference, P < 0.01, ANOVA) compared to controls. Microscopic analysis of murine placentas showed enhanced immunofluorescence of TLR4 in trophoblasts, mainly in the placental labyrinth layer. Also, combined oral infection with C. rectus and P. gingivalis significantly reduced the overall fecundity compared to controls (16.7% vs. 75%, infected vs. non-infected mice respectively, P = 0.03, Kaplan-Meier). The results supported an enhanced placental TLR4 expression after oral infection with periodontal pathogens. The TLR4 pathway has been implicated in the pathogenesis of preterm births; therefore the abnormal regulation of placental TLR4 may give new insights into how maternal periodontitis and periodontal pathogens might be linked to placental inflammation and preterm birth pathogenesis.

Figure 1. Experiment timeline

Female (3-month old) Balb/C mice were pretreated for 4 days with Kanamycin/Ampicillin (50mg/kg-25mg/kg) and then randomly assigned to experimental groups. Mice were infected daily over a 16-week period and mated once/week. When pregnant, mice were sacrificed at embryonic day (E)16.5 and placental tissues were collected and analyzed for TLR4 expression by immunofluorescence (confocal microscopy) followed by RT-PCR. Cr=C. rectus; Pg=P. gingivalis; CMC=carboxymethylcellulose.

Figure 2. Oral infection induces low weight fetuses

Averaged fetal weight values from infected mice were smaller when compared to those from non-infected mice. Columns and bars represent means and standard errors. Cr=C. rectus; Pg=P. gingivalis; *P<0.05, ANOVA.

Figure 3. Oral infection affects murine fecundity

Kaplan Meier Analysis - C. rectus and P. gingivalis infected mice had the lowest fecundity rate (16.7%, HR 0.19[0.041–0.856], P=0.03, Kaplan-Meier) compared to C. rectus (58.3%, Hazard Ratio 0.87[0.310–2.154], P=0.68) and to non-infected mice.

Figure 4. Oral infection induced inflammation and TLR4 expression in murine placentas

Histological and immunofluorescence analysis of murine placentas. 4A: Representative image depicting extensive junctional zone (JZ) along with an increased inflammatory cell infiltrate (arrowheads) in placentas from infected mice. Decidua (D); Bar=50μm. 4B–4C: immunofluorescence of a placenta from the C. rectus and C. rectus/P. gingivalis infected groups respectively. Images represent 3 merged channels (Rhodamine, FITC and DIC) depicting trophoblasts expressing TLR4 from both groups. 4D–E: images depict the expression of Cytokeratin 7 (FITC-green) and TLR4 (Rhodamine-red) in labyrinth trophoblasts from a different C. rectus-infected section; 4F: image illustrates co-localization of Cytokeratin 7 and TLR4 suggesting enhanced expression of TLR4 by labyrinth trophoblasts. 4G-I: images correspond to fluorescence negative controls (no primary antibodies used) for Cytokeratin 7 and TLR4 respectively. Rhod=Rhodamine (red); FITC= Fluorescein isothiocyanat (green); White bars=20μm.

Figure 5. Oral infection with periodontal pathogens increased TLR4 mRNA expression in murine placentas

After normalization, TLR4 mRNA levels were significantly increased in C. rectus group placentas (1.98±0.886 fold difference, P<0.01). Although TLR4 mRNA levels were also higher in the combined infection group, no significant differences were observed when compared to non-infected mice (1.29±0.871, P=0.06). Cr=C. rectus; Pg=P. gingivalis; *P<0.01, ANOVA).